The term “hybrid (or chimeric) protein” refers to a protein obtained as a result of the expression of a recombinant DNA molecule, in which coding arrays of several different genes are bound to one reading frame.
Pneumonia (syn. pulmonic fever) is a common disease of both adults and children and demands conscientious diagnostics and treatment. According to medical statistics, more than one million Russians contract pneumonia annually, and for 5% of patients this disease ends fatally (http://www.who.int/mediacentre/factsheets/fs331/ru/index.html).
The term “pneumonia” refers to a wide range of diseases, each of which possesses its own etiology, pathogenesis, clinical presentation, X-ray characteristics, laboratory data, and specific therapy, can progress as an independent disease or as a sequel of other diseases.
Pneumonia is an inflammatory condition of the human respiratory system. Among the causative agents of pneumonia there are viruses and fungi, but most commonly this disease is caused by bacteria. The most common pathogen that causes bacterial pneumonia is pneumococcus (S. pneumoniae). Examination of pharyngeal microflora has revealed that 5-25% of healthy people are carriers of pneumococcus.
Pneumococci are characterized by a thick polysaccharide capsule that protects the germ against opsonization and subsequent phagocytosis. As the consequence of the fact that the capsule turns to be the main superficial structure of the pneumococci recognized by an immune system, capsule polysaccharide is characterized by the highest variability. Until present, 91 different capsule types of pneumococci have been detected; however, the majority (more than 90%) of invasive pneumococcal diseases is caused by 23 mostly widespread serovars. A high number of immunological variants of capsule polysaccharide turns to be the factor to complicate producing of effective polysaccharide vaccines.
Every human can contract pneumococcal infection, but several risk groups exist, which are susceptible to this disease with higher expectancy. Those are persons 65 years old and older, small infants, persons with certain health disorders, persons with weakened immune system, smokers.
In many cases pneumococcal infection hardly responds to treatment, whereas numerous circulating epidemic pneumococcal stains develop multiple antibiotic resistances. For preventive purposes vaccines are used. According to WHO (World Health Organization) and Russian Respiratory Society, “Vaccination is the only possibility to prevent pneumococcal inflectional pathway”. Currently, FDA (Food and Drug Administration) approved two types of vaccines: pneumococcal conjugate vaccine and pneumococcal polysaccharide vaccine (http://www.nlm.nih.gov/medlineplus/pneumococcalinfections.html). In Russian Federation following vaccines are approved: Pneumo 23 (PPSV23)—a 23-valent pneumococcal polysaccharide vaccine produced by Sanofi Pasteur, France and Prevenar™ (PCV7)—a 7-valent pneumococcal polysaccharide conjugate vaccine produced by Wyeth Pharmaceuticals Inc., USA.
The pneumococcal polysaccharide vaccine (PPSV) is indicated for adults. The majority of adults vaccinated with the PPSV develop resistance to the majority of bacterial strains (the antigens of which are present in the vaccine) 2-3 weeks after inoculation. Among elderly people, children under 2 years, and persons with chronic diseases, vaccination may not lead to generation of persistent immunity, or the immunity to the disease may not develop entirely (http://www.nlm.nih.gov/medlineplus/languages/pneumococcalinfections.html#Russian). Moreover, vaccination can cause such complications as erythema, algesthesis at the injection site, fever, myalgia or severe local reactions (Donalisio M R, Rodrigues S M, Mendes E T, Krutman M. Adverse events after pneumococcal vaccination. J Bras Pneumol. 2007 February; 33(1):51-6).
However, the PPSV turned to be significantly effective only among people with low risk for contracting pneumococcal disease. For children aged under 2 years, simultaneous prophylaxis with antibiotics use during the vaccination is recommended (Bacle A, Diot P, Lemarié E. Anti-pneumococcal vaccine: justifications and results. Rev Pneumol Clin. 1997; 53(3):128-37).
It appeared that the PPSV did not reduce frequency of pneumonia and its fatality rate, but only lowered risk for contracting severe pneumococcal infection. It was also shown that current vaccine did not protect from pneumococcus strains sensitive to penicillin at high concentrations (Pneumococcal vaccine: a second look. Solution for SC or IM injection: pneumococcal vaccine. Prescrire Int. 1998 February; 7(33):16-8). Such results were also reported for people aged over 65 years (Pneumococcal vaccination for elderly subjects: license extension. Still no proof of clinical efficacy. Prescrire Int. 2000 August; 9(48):106-9).
Active ingredient of the PPSV is a mixture of streptococcal polysaccharides of 23 serotypes, which cause up to 90% of invasive diseases with pneumococcal etiology. A polysaccharide is an antigen associated with the T-cell reaction, thus it leads to the development of the short-term immunization solely, without forming immune memory; vaccines containing only above-noted ingredients are ineffective, what has been shown for children aged under 2 years (Greenwood B M et al., Trans R Soc Trop Med Hyg, 1980, 74:756-760; publication of international application WO2010120921 A1, priority date 16 Apr. 2009). The fact that over 90 serotypes of pneumococci exist, as infectious agents of different areas of the world vary, also complicates producing of universal polysaccharide-based vaccine.
Along with polysaccharides of seven pneumococcal serovars, which most frequently cause diseases in children, a conjugate vaccine recommended for children, contains a carrier-protein CRM 197 (diphtheria toxin mutant), which fulfills the role of adjuvant. Due to the presence of adjuvant in the vaccine, this antigen complex is well recognized by T-cells assuring persistent immunity (Schneerson R, Barrera O, Sutton A, Robbins J B. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b polysaccharide-protein conjugates. J Exp Med. 1980 August 1; 152(2):361-76). CRM 197 binds to heparin-binding EGF-like growth factor (HB-EGF). Despite the fact that the toxicity of CRM197 is approximately 106 times lower than that of diphtheria toxin, one should use it carefully, especially at high doses (Takuya Kageyama, Minako Ohishil, Shingo Miyamoto, Hiroto Mizushima, Ryo Iwamoto and Eisuke Mekada. Diphtheria Toxin Mutant CRM197 Possesses Weak EF2-ADP-ribosyl Activity that Potentiates its Anti-tumorigenic Activity. Received Apr. 16, 2007. Accepted May 2, 2007). Therewithal, vaccination can cause following complications: erythema, dermatodynia and induration at the injection site, rise in body temperature to 38-39° C. (100.4-102.2° F.) as well as restlessness, sleepiness and loss of appetite. Moreover, it is recommended to vaccinate children in the age of 2 years 4 times, in the age of 2 to 5 years—depending on the age of the child. In total, at least four doses are needed to vaccinate a child, which is expensive and unsafe.
A vaccine is known containing two main components: a superficial polysaccharide of N. meningitidi and a PsaA-protein of S. pneumoniae (PspA-protein can also be used) (publication of international application WO2010120921 A1, priority date 16 Apr. 2009). Taking into consideration the inability to develop immune memory to polysaccharides, we deem usage of the superficial polysaccharide of N. meningitidi as a vaccine component to be inappropriate. The usage of the PsaA- and PspA-proteins in its turn is reasonable: those are pneumococcal superficial antigens, which are capable of inducing both immune response and immune memory.
The effect of a vaccine to be patented is based on the induction of strong immune response and the subsequent development of the immune memory. Therefore the central position of our invention is occupied not by polysaccharides, but by pneumococcal proteins obtained using methods based on molecular biology and recombinant DNA. According to data in literature, the most promising protein-based antigens are three superficial proteins: PsaA, PspA, Spr1895.
PsaA-protein is also considered to be a promising immunogen by the authors of the international application publicized as WO2004102199 A2, priority date 16 May 2003. The authors mention this and several other proteins (SlrA—lipoprotein rotamase, IgA1—protease, PpmA—streptococcal maturation protein) or their compounds as the basis for the vaccine creation. However, we consider approach of using other surface proteins of S. pneumoniae—beside PsaA, PspA and Spr1895—to be more promising.
A chimeric protein containing PsaA and B subunit of cholera toxin with adjuvant function is described (Arêas A P, Oliveira M L, Miyaji E N, Leite L C, Aires K A, Dias W O, Ho P L. Expression and characterization of cholera toxin B-pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice. Biochem Biophys Res Commun. 2004 Aug. 13; 321(1):192-6). However, we believe it is less safe to use cholera toxin or its compounds—compared to flagellin—as a vaccine component, whereas human organism is highly sensitive to cholera toxin, so that even 8 μg of toxin can cause strong diarrhea.
Studies have shown that it is reasonable to use PsaA- and PspA-proteins as basis for a pneumococcal vaccine creation. The PsaA-protein is highly conserved among different pneumococcal serotypes and provides bacterial adhesion and its virulence (Berry A M, and Paton J C: Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae. Infect Immun 1996; 64:5255-5262). It was shown that PsaA specific antibodies possess a cross-linking activity towards all serotypes of S. pneumonia. 
PspA is a choline-binding surface antigen that inhibits complement-independent phagocytosis, binds to lactoferrin, and prevents lactoferrin-independent elimination of bacterial cells (Hammerschmidt S, Bethe G, Remane P H, Chhatwal G S (1999) Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae. Infect Immun 67:1683-1687). In this paper the PsaA-protein is mentioned to be promising for producing of the vaccine. The authors have also shown a range of protein candidates for producing vaccines against pneumococcal infection; however, we consider it to be reasonable to use two particular proteins: PsaA and PspA, considering their conservatism among serotypes of pneumococci and several other bacteria.
One of the main components of the offered vaccine is a Spr1895-protein coded by the gene of a phosphate-binding protein—phosphate ABC transporter. This protein is essential for bacterial viability and constant, which makes it the component of choice for producing of the pneumococcal vaccine against S. pneumonia-caused diseases.
In the present invention, flagellin protein FliC fulfills function of adjuvant. Due to its interaction with Toll-like receptor-5 (TLR-5), FliC stimulates maturation of macrophages and dendritic cells, which results in development of the immune response (Mc Dermott P. F. High-affinity interaction between Gram-negative flagellin and a cell surface polypeptide results in human monocyte activation. Infect. Immun. —2000.—V. 68. —p.: 5525-5529; Means T. K. et al. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J. Immunol.—2003.—V. 170. —p.: 5165-5175).
Currently, flagellin is considered to be one of the most promising and well characterized new generation adjuvants. The results of the research show that recombinant proteins injected together with flagellin possess increased immunogenic and antigenic characteristics. Reactions thereto are recorded in shorter terms and result in stronger cell-mediated and humoral immune response (Balaram, 2008).
In the present invention it is shown that flagellin components can function as adjuvant. Two receptor-activating domains were discovered in the terminal regions of flagellin (aa 79-117 and aa 408-439) (Tonyia, 2001).
Thus, the approach using definite components of flagellin is reasonable (FliC domain 1, FliC domain 2).
TLR-5 is expressed on the surface of the cells of the innate immunity, epithelial, and endothelial cells (Sebastiani G. et al. Cloning and characterization of the murine Toll-like receptor 5(Tlr5) gene: sequence and mRNA expression studies in Salmonella-susceptibleMOLF/Eimice. Genomics.—2000.—V. 64.—p.230-240; Zarember K. A. and Godowski P. J. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J. Immunol.—2002.—V.168.—p.554-561; Delneste, 2007). In view of this, it is reasonable to use mucosa for immunization, which considerably facilitates transport of an immunogen.
In such a way, the invention described in WO2004102199 A2 and construct CTB-PsaA described in article mentioned above can be referred to to be the most akin to the present invention. However, in the mentioned international application it is proposed to use proteins or their functional units as separate vaccine components. Our invention is yet based on a chimeric protein that except for its specific S. pneumoniae protein constituent also includes an adjuvant. Therefore, the prototype of the present invention is a chimeric protein described by the Brazil authors.
The chimeric protein CTB-PsaA includes PsaA of the 6B serotype of S. pneumoniae (289 aa) as well as the B-subunit of cholera toxin functioning as adjuvant (Arêas A P, Oliveira M L, Miyaji E N, Leite L C, Aires K A, Dias W O, Ho P L. Expression and characterization of cholera toxin B-pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice. Biochem Biophys Res Commun. 2004 August 13; 321(1):192-6). However, it is more beneficial and easy—for provision of the correct molecular folding—to use a protein fragment, the antigenic determinant instead of the full-sized protein. Exactly this approach—usage of the most immunogenic protein fragments—was taken for creation of this invention. We do also consider usage of cholera toxin or its components as a vaccine component to be less safe in comparison to flagellin, since people are highly sensitive to the cholera toxin, and even 8 μg toxin can cause strong diarrhea. As for the method for producing of this chimeric protein, the gene encoding PsaA-protein was amplified separately from the plasmid, in which it was later inserted, followed by cloning via restriction-sites in a vector containing the CTB-encoding gene. In our case we used synthesis of the full-sized gene coding for the chimeric protein.